Bacteriophage preparations and methods of use thereof

ABSTRACT

Disclosed herein are purified bacteriophage preparations that effectively lyse a plurality of  C. perfringens  strains. In one embodiment, a purified bacteriophage preparation includes four or more  C. perfringens -specific bacteriophage, wherein each bacteriophage has lytic activity against at least five  Clostridium  species strains. In another embodiment, the purified bacteriophage preparation includes five or more  C. perfringens -specific bacteriophage.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. patent application Ser. No.12/334,863 filed Dec. 15, 2008 which claims priority to U.S. ProvisionalApplication Ser. No. 61/013,325 filed Dec. 13, 2007, which isincorporated in its entirety by reference herein.

BACKGROUND

Antibiotic use enhances the growth of healthy domesticated poultry andlivestock. Although extensive bans and restrictions have not beenimplemented in the United States as they have in the E.U. and othercountries, pressure for antibiotics alternatives has increased due toconcerns of increasing antibiotic resistance among food borne bacteria.Banning or markedly reducing the agricultural and farm-veterinary use ofantibiotics may have a profound negative impact on the safety of foodsand on the treatment of sick flocks or herds of domesticated livestock,however. Thus, effective, safe and environmentally friendlyalternative(s) to antibiotics are needed to address these concerns andneeds.

Viruses that kill bacteria were first identified in the early part ofthe 20^(th) century by Frederick Twort and Felix d'Herelle who calledthem bacteriophages or bacteria-eaters (from the Greek phago meaning toeat or devour). Because of their remarkable antibacterial activity,phages were used to treat disease of economically importantanimals/domesticated livestock almost immediately after their discovery,and therapeutic applications for humans closely followed. However, withthe advent of antibiotics, phage therapy gradually fell out-of-favor inthe United States and Western Europe, and virtually no subsequentresearch was done on the potential therapeutic application of phages forbacterial diseases of humans or animals. The emergence ofantibiotic-resistance in bacteria, however, has rekindled interest intherapeutic bacteriophages. Phage therapy may have a positive impact onhuman health by improving the safety of foods in the U.S.A. andelsewhere, and by helping to reduce safely the use of antibiotics inagribusiness.

Among the bacteria that cause significant morbidity and mortality inchickens, C. perfringens is one of the most notorious pathogens. Inchickens, C. perfringens infections are often manifested as necroticenteritis that occur later in the production cycle, often following acoccidial infection or other insult to the gastrointestinal tract. It isthus desirable to develop bacteriophage preparations suitable to reducemorbidity and mortality in chickens.

SUMMARY

Disclosed herein are purified bacteriophage preparations thateffectively lyse a plurality of C. perfringens strains. In oneembodiment, a purified bacteriophage preparation comprises four or moreC. perfringens-specific bacteriophage, wherein each bacteriophage haslytic activity against at least five C. perfringens strains. In anotherembodiment, the purified bacteriophage preparation comprises five ormore C. perfringens-specific bacteriophage.

In another embodiment, a method of reducing chicken mortality due to C.perfringens infection comprises administering a purified bacteriophagepreparation comprising four or more C. perfringens-specificbacteriophage, wherein each bacteriophage has lytic activity against atleast five C. perfringens strains.

In another embodiment, a method of selecting a C. perfringens hoststrain suitable to propagate bacteriophage from a plurality of teststrains comprises microbiologically confirming one test strain from theplurality of test strains as a C. perfringens species to produce aconfirmed strain; associating the confirmed strain with a poultrydisease to produce a disease-associated strain; and applying one or moreadditional selective criterion to the disease-associated strain selectedfrom minimal antibiotic resistance and absence of animal-virulencemarkers other than those for C. perfringens to produce the C.perfringens host strain suitable to propagate bacteriophage.

In yet another embodiment, a method of producing a bacteriophagecocktail comprises mixing four or more C. perfringens-specificbacteriophage, wherein each bacteriophage has lytic activity against atleast five C. perfringens strains.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a phylogenetic analysis of C. perfringens strains.

FIG. 2 is a dendrogram portraying the genetic diversity of various C.perfringens strains based on SmaI-digested PFGE patterns of C.perfringens DNA.

FIG. 3 shows phage plaques produced by representative bacteriophageinfecting C. perfringens strain Cp 42.

FIG. 4: Pulsed-field gel electrophoresis analysis of undigested phageDNA isolated from each monophage. Lane M, Low Range PFG Marker (NEB);lane 1, CPAS-7; lane 2, CPAS-15; lane 3, CPAS-16; lane 4, CPLV-42; lane5, CPTA-12; lane 6, CPAS-37.

FIG. 5 shows XmnI digested phage DNA from each monophage. Lane M,GeneRuler DNA Ladder Mix (Fermentas); lane 1, CPLV-42; lane 2, CPAS-7;lane 3, CPAS-15; lane 4, CPTA-37; lane 5, CPAS-12; lane 6, CPAS-16.

FIG. 6 shows structural protein profiles for each C. perfringensmonophage. Lane 1, CPLV-42; lane 2, CPAS-12; lane 3, CPAS-7; lanes 4,CPAS-16; lane 5, CPAS-15; lane 6, CPTA-37.

FIG. 7 shows electron micrographs of C. perfringens bacteriophages.

The above-described and other features will be appreciated andunderstood by those skilled in the art from the following detaileddescription, drawings, and appended claims.

DETAILED DESCRIPTION

Disclosed herein are purified bacteriophage preparations thateffectively lyse a plurality of C. perfringens strains. Lysis ofparticular strains is demonstrated by the drop-on-lawn method, which isstandard in the art. The bacteriophage preparations are suitable toreduce morbidity and mortality in chickens.

In one embodiment, a purified bacteriophage preparation comprises fouror more C. perfringens-specific bacteriophage, wherein eachbacteriophage has lytic activity against at least five C. perfringensstrains. In another embodiment, the purified bacteriophage preparationcomprises five or more C. perfringens-specific bacteriophage.

In one specific embodiment, the bacteriophage preparation comprisesCPAS-12 (accession number PTA-8479), CPAS-15 (accession numberPTA-8480), CPAS-16 (accession number PTA-8481) and CPLV-42 (accessionnumber PTA-8483).

In another embodiment, the bacteriophage preparation consistsessentially of CPAS-12, CPAS-15, CPAS-16 and CPLV-42. In yet anotherembodiment, the bacteriophage preparation consists of CPAS-12, CPAS-15,CPAS-16 and CPLV-42.

In another specific embodiment, the bacteriophage preparation comprisesCPAS-7 (accession number PTA-8478), CPAS-12, CPAS-15, CPAS-16 andCPLV-42. In another embodiment, the bacteriophage preparation consistsessentially of CPAS-7, CPAS-12, CPAS-15, CPAS-16 and CPLV-42. In yetanother embodiment, the bacteriophage preparation consists of CPAS-7,CPAS-12, CPAS-15, CPAS-16 and CPLV-42.

In one embodiment, the C. perfringens strains are ATCC strain 25768,ATCC strain 3624, ATCC strain 9856, ATCC strain 3628, ATCC strain 13124,ATCC_ strain PTA-8495, NRRL strain B-50143, NRRL strain B-50144, NRRLstrain B-50145, and combinations comprising one or more of the foregoingstrains. In a specific embodiment, the at least five C. perfringensstrains comprise ATCC strain 3624 and ATCC strain 9856.

In one embodiment, the bacteriophage preparations are characterized bytheir specificity and effectiveness against C. perfringens strains. Inone embodiment, the purified bacteriophage preparation lyses greaterthan or equal to 85% of at least 40 screened C. perfringens strains,wherein the bacteriophage preparation is incapable of infecting at least10 strains of E. coli, L. monocytogenes, S. enterica, and P. aeruginosa.In another embodiment, the purified bacteriophage preparation lysesgreater than or equal to 85% of at least 45 screened C. perfringensstrains. In yet another embodiment, each of the individual C.perfringens-specific bacteriophage lyses 15% to 90% of the screened C.perfringens strains.

The term “purified” in reference to a bacteriophage or bacteriophagepreparation does not require absolute purity (such as a homogeneouspreparation). Instead, it is an indication that the bacteriophage orbacteriophage preparation is relatively more pure than in the naturalenvironment (compared to the natural level, this level should be atleast 2-5 fold greater, e.g., in terms of mg/mL). Purification isaccording to any method known to those of ordinary skill in the art thatwill result in a preparation of bacteriophage substantially free fromother nucleic acids, proteins, carbohydrates, lipids, or subcellularorganelles. Individual bacteriophage may be purified to electrophoretichomogeneity. Purification of at least one order of magnitude, preferablytwo or three orders, and more preferably four or five orders ofmagnitude is expressly contemplated.

Purity of phage stocks can be determined by pulsed-field gelelectrophoresis (PFGE) of uncut DNA. In one embodiment, approximately100-200 ng of the phage DNA is electrophoresed in a 1 SeaKem GoldAgarose (Cambrex, Rockland, Me.) gel with 0.5× Sodium boric acid (1×SB:10 mM sodium hydroxide pH adjusted to 8.5 with boric acid) buffer at 14°C. in a CHEF Mapper XA PFGE apparatus (Bio-Rad Laboratories, Hercules,Calif.). The run time is 12 hours with a voltage of 6 V/cm and alinearly ramped pulse time of 0.06 s to 8.53 seconds. The gels warestained with ethidium bromide and visualized with UV light.

In one embodiment, endotoxin levels for lots of phage produced aredetermined by a Limulus amoebocyte lysate (LAL) assay using achromogenic assay (QCL-1000 Kit, BioWhittaker, Walkersville, Md.)following the manufacturers' recommendations. A standard curve with E.coli endotoxin (supplied with kit) ranging from 0.1 to 1.0 endotoxinunits (EU) per milliliter endotoxin is constructed for each assay byplotting the optical density at 405 nm (OD₄₀₅) versus EU/ml. Phagesamples are assayed diluted in sterile water (USP, Baxter, Deerfield,Ill.) and the absorbance at 405 nm was measured with a μQuant (Bio-TekInstruments, Inc., Winooski, Vt.) microplate reader. Endotoxinconcentrations for each sample are calculated by linear regression fromthe standard curve. Standards and samples are analyzed in triplicate.

In one embodiment, carbohydrate content for all lots of phage producedis determined by an anthrone method. A standard curve with glucoseranging from 10 to 250 μg/ml is constructed for each assay by plottingthe optical density at 625 nm (OD₆₂₅) versus μg/ml of glucoseconcentration. Phage samples are assayed in PBS and the absorbance at625 nm is measured with a μQuant (Bio-Tek Instruments, Inc.) microplatereader. Carbohydrate concentrations for each sample are calculated bylinear regression from the standard curve. Standards and samples areanalyzed in duplicate.

In one embodiment, the total protein content for lots of phage producedis determined by a bicinchoninic acid (BCA) assay using a colorimetricassay (BCA™ Protein Assay Kit, Pierce, Rockford, Ill.) following themanufacturers' recommendations. A standard curve with bovine serumalbumin (BSA, supplied with kit) ranging from 20 to 2,000 μg/ml proteinis constructed for each assay by plotting the optical density at 562 nm(OD₅₆₂) versus μg/ml BSA. The absorbance at 562 nm is measured with aμQuant (Bio-Tek Instruments, Inc.) microplate reader. Total proteinconcentrations for each sample are calculated by linear regression fromthe standard curve. Standards and samples are analyzed in triplicate.

The term “strain” means bacteria or bacteriophage having a particulargenetic content. The genetic content includes genomic content as well asrecombinant vectors. Thus, for example, two otherwise identicalbacterial cells would represent different strains if each contained avector, e.g., a plasmid, with different phage open reading frameinserts.

By “treatment” or “treating” is meant administering a bacteriophagepreparation for prophylactic and/or therapeutic purposes. The term“prophylactic treatment” refers to treating an animal that is not yetinfected but is susceptible to or otherwise at risk of a bacterialinfection. The term “therapeutic treatment” refers to administeringtreatment to an animal already suffering from infection.

The term “bacterial infection” means the invasion of the host organism,animal or plant, by pathogenic bacteria. This includes the excessivegrowth of bacteria which are normally present in or on the body of theorganism, but more generally, a bacterial infection is any situation inwhich the presence of a bacterial population(s) is damaging to a hostorganism. Thus, for example, an organism suffers from a bacterialinfection when excessive numbers of a bacterial population are presentin or on the organism's body, or when the effects of the presence of abacterial population(s) is damaging to the cells, tissue, or organs ofthe organism.

The terms “administer”, “administering”, and “administration” refer to amethod of giving a dosage of a bacteriophage preparation to an organism.Suitable methods include topical, oral, intravenous, transdermal,intraperitoneal, intramuscular, or intrathecal. The preferred method ofadministration varies depending on various factors, e.g., the componentsof the bacteriophage preparation, the site of the potential or actualbacterial infection, the bacterium involved, and the infection severity.

In the context of treating a bacterial infection a “therapeuticallyeffective amount” or “pharmaceutically effective amount” indicates anamount of a bacteriophage preparation which has a therapeutic effect.This generally refers to the inhibition, to some extent, of the normalcellular functioning of bacterial cells that render or contribute tobacterial infection.

The dose of bacteriophage preparation that is useful as a treatment is a“therapeutically effective amount”. Thus, as used herein, atherapeutically effective amount means an amount of a bacteriophagepreparation that produces the desired therapeutic effect as judged byclinical trial results. This amount can be routinely determined by oneskilled in the art and will vary depending on several factors, such asthe particular bacterial strain involved and the particularbacteriophage preparation used.

In one embodiment, a bacteriophage preparation optionally includes oneor more pharmaceutically acceptable excipients. In one embodiment, theexcipient is a water-conditioning agent, for example agents suitable forwater dechlorination and/or phage stabilization. Such agents areinnocuous to the bacteriophage cocktail, but when added prior to orsimultaneously with C. perfringens bacteriophage or bacteriophagecocktails, act to dechlorinate municipal levels of chlorine, which ifuntreated would kill or significantly reduce the viability of thebacteriophage or bacteriophage cocktail. Exemplary water-conditioningagents include amino acids and/or salts which help to normalize the pHand ionic balance of the bacteriophage cocktail, when added to diversewater sources used for the animal's drinking and for phage delivery. Inone embodiment, the water-conditioning agent is a 50 mMcitrate-phosphate-thiosulfate (CPT) buffer, comprising about 40 mgsodium thiosulfate, 6.0 gm disodium phosphate (anhydrous), 1.1 gm citricacid (anhydrous) per liter of deionized water, pH 7.0. By includingwater-conditioning agents in the cocktail or adding separately to thetreatment water, the water-conditioning agents act to both stabilize andprotect the bacteriophage cocktail in a commercial preparation suitablefor routine field use.

A method of producing a bacteriophage cocktail comprises mixing four ormore C. perfringens-specific bacteriophage, wherein each bacteriophagehas lytic activity against at least 5 C. perfringens strains. In anotherembodiment, a method of producing a bacteriophage cocktail comprisesmixing five or more C. perfringens-specific bacteriophage, wherein eachbacteriophage has lytic activity against at least 5 C. perfringensstrains

Once C. perfringens cocktail have been selected, further testing can beemployed to refine the specificity of the cocktail. In one embodiment, abacteriophage cocktail is tested against additional C. perfringensstrains which have antibiotic resistance genes to test the phagecocktail against antibiotic-resistant strains and evaluate thecocktail's potential for use in the field against antibiotic-resistantClostridia. In another embodiment, a bacteriophage cocktail is testedagainst C. perfringens strains derived from animal species other thanchickens to further evaluate and define the host range of the strain orstrains to include additional animal species (e.g., swine, cattle,turkey, sheep, exotics, dogs, cats, and the like) In another embodiment,a bacteriophage cocktail is tested against Clostridium of differentspecies (i.e., other than C. perfringens), which will further define therange of effectiveness of the phage cocktail. In yet another embodiment,a bacteriophage cocktail is tested against additional gram-positivebacteria (e.g., both reference strains and animal-associated types,aerobic and anaerobic) to further evaluate and define host range. Inanother embodiment, a bacteriophage cocktail is tested againstadditional gram-negative and gram-variable microorganisms to furtherevaluate host range. In another embodiment, a bacteriophage cocktail istested for pre-conditioning or additive formulations, using differentlevels of stabilizing and dechlorinating water-conditioning agents, foroptimally maintaining the viability of the phage cocktail under a widerange of water types and chlorination levels as may be expected in fieldusage conditions.

In another embodiment, the method further comprises testing thepotential of the bacteriophage cocktail for the development of intrinsicphage resistance in the target host. Testing includes challenging testC. perfringens host strains with individual and/or combinedbacteriophage cocktail phage over several cycles, and ascertaining therate of resistance development toward the individual phages as well asthat of the combination(s) of phages. It is anticipated that thecombination bacteriophage cocktail will have significantly lessdevelopment of resistance against a given individual host strain. Anoptimum combination of bacteriophages may be further elucidated usingknown mathematical optimization techniques or software packages(Box-Hunter, Latin Squares, Taguchi, Simplex, etc.) as applied to thebacteriophage resistance data generated from such experimentation.

Advantages of bacteriophage therapy include high bactericial activity,high selectivity permitting targeting of specific pathogens whileleaving desirable bacterial flora intact, specificity for prokaryoticcells, and environmental benignity. In livestock and poultryapplications, bacteriophage have the advantage of specificity thatshould not select for phage-resistance in non-target bacterial species,the possible emergence of resistance against phages will not affect thesusceptibility of the bacteria to antibiotics used to treat humans, and,unlike antibiotics, phage preparations can readily be modified inresponse to changes in bacterial pathogen populations or susceptibility.

The poultry and livestock industries use antibiotics for three mainpurposes: (i) prophylactically, to prevent disease in flocks, herds,etc., (ii) to treat sick livestock, and (iii) to improve digestion andutilization of feed, which often results in improved weight gain.Antibiotics used in the latter setting often are referred to as“growth-promoting antibiotics” or GPAs. Most GPAs are not commonly usedin human medicine, and they are usually administered, in small amounts,to poultry and other livestock via food. Bacteriophages can effectivelyreplace and/or reduce the use of antibiotics in all three of theabove-mentioned settings.

Among the bacteria that cause significant morbidity and mortality inchickens, C. perfringens is one of the most notorious pathogens. Inorder to identify effective bacteriophage for a genetically diversepopulation of C. perfringens strains, isolates of C. perfringens werefirst identified. In order to identify effective bacteriophage, it isuseful to identify C. perfringens strains that affect poultry at variouslocations within the United States. As shown herein, forty-one strainsof C. perfringens were isolated from various sources and characterizedby pulsed-field gel electrophoresis (PFGE) typing. (FIG. 1) Among the 35strains subjected to PFGE, phylogenetic analysis showed that thesestrains clustered into 15 heterogenic groups. (FIG. 2) Among these 15PFGE types, P6 is the predominant type (10 strains) followed by P4 (6strains). Additional C. perfringens strains may be obtained frompublicly available collections.

One important factor in the identification of bacteriophage is theselection of C. perfringens strains suitable for their identification.In one embodiment, a method of selecting a C. perfringens host strainsuitable to propagate bacteriophage from a plurality of test strains,comprises microbiologically confirming one test strain from theplurality of test strains as a C. perfringens species to produce aconfirmed strain; associating the confirmed strain with a poultrydisease to produce a disease-associated strain; applying one or moreadditional selective criterion to the disease-associated strain selectedfrom minimal antibiotic resistance and absence of animal-virulencemarkers other than those for C. perfringens to produce the C.perfringens host strain suitable to propagate bacteriophage. In oneembodiment, the selection criterion is minimal antibiotic resistance andthe antibiotic resistance is tetracycline, ampicillin, tylosin,erythromycin, lincomycin, chloramphenicol or other drug resistance. Theselection of strains absent from antibiotic resistance minimizes thepotential transduction of plasmid or chromosomal-borne antibioticresistance genes, into the subsequent bacteriophage cocktail genomes.The advantage of this applied criterion, is to in advance, limit anypotential resistance genes in a bacteriophage cocktail preparation. Theselective criterion used for these phage cocktail host strains, are aunique extension of a unique library of C. perfringens strains, combinedwith microbiological knowledge of antibiotic resistance, along withskills in running antibiotic susceptibility tests to ascertain theresistance profiles of the submitted host strains.

Six novel bacteriophages of the Siphoviridae or Myoviridae families thatinfect Clostridium perfringens were isolated from environmental water orsewage sources. Phage are characterized, for example, at both theprotein and nucleic acid level. The optimal host strain for propagationof each bacteriophage is identified and all phage are preferablynegative for endogenous phage. In addition, each bacteriophage ischaracterized by PFGE, RAPD, SDS-PAGE, and other approaches. Stocks ofall six monophages and their respective host strains are made for use incharacterization and production of each phage.

The C. perfringens-specific monophages are capable of specificallyinfecting C. perfringens strains, and are not capable ofinfecting/growing on E. coli, L. monocytogenes, S. enterica, and P.aeruginosa. As used herein, the term C. perfringens-specific refers tobacteriophage and bacteriophage preparations that are capable ofinfecting a plurality of C. perfringens strains and are incapable ofinfecting at least 10 strains of E. coli, L. monocytogenes, S. enterica,and P. aeruginosa.

Six bacteriophages that infect Clostridium perfringens are sequenced.(SEQ ID NOs:1-6) Five of the six phages are sequenced, and eachpredicted open reading frame is identified in each genome. Each of thepredicted genes was annotated. None of the 17 undesirable genes (Table5.1.1) is found in the genomes of any of the five phages for whichsequences were available.

Two phage cocktails, INT-401 (CPAS-7, CPAS-12, CPAS-15, CPAS-16, andCPLV-42) and INT-402 (CPAS-12, CPAS-15, CPAS-16, CPLV-42), are preparedfrom five of the six monophages isolated. Both cocktails are effectivein killing greater than 85% of the 46 C. perfringens strains screened.INT-401 was selected for use in proof-of-principle efficacy studiesdesigned to determine the prevention of necrotic enteritis in C.perfringens challenged broiler chickens.

Oral Gavage of Test Article (INT-401 phage cocktail) to birds on the dayof challenge (Day 14) and for the next four days significantly reducedmortality due to NE. Growth performance in this group was numericallyequivalent to the non-challenged control, and appeared to be bettercompared to the challenged, but phage-untreated chickens. Given the factthat many chickens are naturally colonized with C. perfringens, thelatter observation warrants further elucidation, to better examine thepossible growth performance-enhancing benefits of the phage preparation.

Two of the three “in ovo” treatments had numerically reduced NEmortality (9.6 and 14.8%) when compared to the Challenged control(25.9%).

Oral Gavage of Test Article prior to challenge, or spray of Test Articleto chicks at the hatchery, was ineffective in preventing NE mortalitydue to C. perfringens challenge.

The results of the studies herein suggest that C. perfringens-specificphage preparation can be effective in significantly reducing chickenmortality due to C. perfringens infections in chickens such as thosecausing necrotic enteritis when administered shortly after the bacterialchallenge. Further dosing- and delivery-optimization studies arewarranted, together with further fine-tuning of the product for theoptimal efficacy.

Exemplary means of administration of the bacteriophage preparations areoral administration, intramuscular injection, subcutaneous injection,intravenous injection, intraperitoneal injection, eye drop, nasal spray,and the like. When the subject to be treated is a bird, the bird may bea hatched bird, including a newly hatched (i.e., about the first threedays after hatch), adolescent, and adult birds. Birds may beadministered the vaccine in ovo, as described in U.S. Pat. No. 4,458,630to Sharma, for example, incorporated herein by reference.

In one embodiment, the bacteriophage preparation is administered in ananimal feed such as poultry feed. The bacteriophage preparation isprepared in a number of ways. For instance, it can be prepared simply bymixing the different appropriate compounds to produce the bacteriophagepreparation. The resulting bacteriophage preparation can then be eithermixed directly with a feed, or more conventionally impregnated onto acereal-based carrier material such as milled wheat, maize or soya flour.Such an impregnated carrier constitutes a feed additive, for example.

The bacteriophage preparation may be mixed directly with the animalfeed, or alternatively mixed with one or more other feed additives suchas a vitamin feed additive, a mineral feed additive or an amino acidfeed additive. The resulting feed additive including several differenttypes of components can then be mixed in an appropriate amount with thefeed. It is also possible to include the bacteriophage preparation inthe animal's diet by incorporating it into a second (and different) feedor drinking water which the animal also has access to. Accordingly, itis not essential that the bacteriophage preparation is incorporated intothe usual cereal-based main feed of an animal.

In one embodiment, included are methods of identifying an optimizedfield delivery modes and conditions for phage cocktail applications. Inone embodiment, an optimized administration condition is wateradministration, for up to 3 days at temperatures up to 50° C.

The bacteriophage preparation can be used for a wide variety of animals,but use of the bacteriophage preparation is particularly preferred indomestic animals and farm livestock. Animals which may in particularbenefit from the bacteriophage preparation include poultry (such aschickens, turkeys, ducks and geese), ruminants (such as cattle, horsesand sheep), swine (pigs), rodents (such as rabbits) and fish. Thebacteriophage preparation is particularly useful in broiler chickens.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Characterization of Clostridium perfringens Isolates

Media:

Brain Heart Infusion (BHI) broth or BHI agar was used to grow allisolates. All media were obtained from EMD Chemicals, Gibbstown, N.J.

Microorganisms:

Forty-two C. perfringens strains were employed. One strain (Cp 20) didnot grow and was excluded from further analysis. As part of thecollection process, isolates were checked for purity and frozen at −80°C. in 30% glycerol. Most of the work was performed in an anaerobicchamber (Plas-Labs, Inc. Lansing, Mich.), that contained a 90% N₂-5%H₂-5% CO₂ atmosphere.

Bacteriophage:

All bacteriophages were isolated from environmental water sources.

Phage Isolation:

Samples of water collected for the isolation of phage were mixed with10×BHI broth, inoculated with a single C. perfringens strain of interestand incubated anaerobically at 37° C. overnight. The samples werecentrifuged (8,000×g, 10 min) to remove the bacterial cells and sterilefiltered (0.22 μm Stericup™, Millipore, Bedford, Mass.). Filtrates wereserially diluted in BHI broth and titered using the soft-agar overlaymethod. Briefly, dilutions of each filtrate were mixed with log-phasebacterial culture, incubated at 37° C. for 10 minutes, molten soft-agaradded, poured onto BHI agar plates and incubated anaerobically at 37° C.overnight. Individual plaques were picked from the overlay plates andtitered a second time as an initial step in ensuring that each phage waspure.

Screening for Endogenous Phage:

Clostridium perfringens strains used for propagating the phages werescreened for endogenous bacteriophage by the drop on lawn method. Liquidcultures of the host strains were grown overnight, centrifuged (9,500×g,5 minutes) to remove the bacteria and filtered through a 0.22 gm syringefilter (Millipore). The same strains were grown in BHI broth to an OD₆₀₀of 0.1-0.3. Two hundred microliters of each screening strain was mixedwith molten soft-agar and poured onto a BHI agar plate. After thesoft-agar hardened 10 μl of each host strain filtrate was spotted ontothe plates with the screening strains. Lytic activity was observed afterovernight anaerobic incubation at 37° C.

Clostridium perfringens Host Strain Typing:

The 41 C. perfringens strains received were typed by PFGE using theNational Molecular Subtyping Network (PulseNet) standard protocol.Clostridium perfringens strains were grown on BHI agar overnightanaerobically at 37° C. and suspended in 75 mM NaCl-25 mM EDTA (pH 8.0)(CSB) buffer to an OD₆₁₀ of 1.3-1.4. The bacterial cells were embeddedin 1.2% SeaKem® Gold Agarose (Cambrex, Rockland, Me.) by mixing equalvolumes (0.4 mL) of the cell suspension and melted agarose made in TEbuffer. Plugs were made in 1.5-mm thick molds (Bio-Rad Laboratories,Hercules, Calif.) and solidified at 4° C. The cells were lysed byincubation in lysis buffer (50 mM Tris-HCl [pH 8.0], 50 mM EDTA [pH8.0], 1% N-laurylsarcosine, and proteinase K [1 mg/ml]) at 55° C.overnight. The plugs were washed at 54° C. with shaking three times for15 minutes each in sterile water and then three times in TE buffer. Theplugs were stored at 4° C. in TE buffer.

Plugs were equilibrated with restriction endonuclease buffer at 25° C.overnight. The plugs were digested with SmaI (New England Biolabs,Beverly, Mass.) according to the manufacturers' recommendationsovernight. Restriction fragments were separated by electrophoresisthrough a 1% agarose gel in 0.5× Tris-borate-EDTA (10× TBE, EMDChemicals) with 1 mM thio-urea at 14° C. in a CHEF Mapper XA PFGEapparatus (Bio-Rad Laboratories). The run time was 20 hours with avoltage of 6 V/cm and a linearly ramped pulse time of 0.4 seconds to 40seconds. The size range analyzed was 40-1,400 kilobases.

Data Handling & Analysis:

A large zone of clearing (lytic activity) produced on lawns of any ofthe C. perfringens strains where the culture filtrate was applied wereconsidered positive for endogenous phage.

Host Strain Typing:

Analysis of the PFGE banding patterns was done with Quantity One (ver.4.41) and Molecular Analyst Fingerprinting (ver. 1.6) software (Bio-RadLaboratories) to determine the genetic relatedness of the strains. Thedendrogram was constructed by the UPGMA algorithm with a 4% tolerancefor fragment shifts. Isolates were considered closely related if theirPFGE patterns differed by less than three fragments when digested withthe restriction endonuclease SmaI.

PFGE Results:

Forty-one strains of C. perfringens were isolated from various sourcesand characterized by pulsed-field gel electrophoresis (PFGE) typing.(Table 1) Among the 35 strains (one strain did not grow and six were nottype-able due to nuclease problems) subjected to PFGE, phylogeneticanalysis showed that these strains clustered into 15 heterogenic groups.(FIG. 1) Among these 15 PFGE types, P6 is the predominant type (10strains) followed by P4 (6 strains). C. perfringens strain Cp27 has ATCCaccession number PTA-8495.

TABLE 1 Clostridium perfringens isolates Intralytix Alpharma IsolationPathogenic PFGE ID ID* Year Source Location (Yes/No) Comment Type Cp 17998 B 1995 Roney Canada Yes P1 Cp 2 UAZ 75 — — — — P2 Cp 3 Wallers 1993— IL Yes P3 Cp 4 Pennington 1993 — IL Yes P4 Cp 5 96-7413 1996 Roney ALYes NT Cp 6 UAZ 74 — — — — P5 Cp 7 Warren 1993 — IL Yes P6 Cp 8 AU1 1996— AL Yes Gangrenous P7 Dermatitis Cp 9 95-949 1995 Fitz-Coy East CoastYes NT Cp 10 M1 2000 Fitz-Coy East Coast Yes P8 Cp 11 Harmes 1993 — ILYes P3 Cp 12 94-5223 1994 Thayer GA Yes P6 Cp 13 D00-20250 2000 Fitz-CoyMN Yes NT Cp 14 UDE 95-1377 1995 Fitz-Coy DE Yes P9 Cp 15 95-1046 1995Fitz-Coy DE Yes Gall Bladder NT Cp 16 F96-01993 1996 Fitz-Coy CA Yes P6Cp 17 UAZ 257 — — — — P10 Cp 18 94-5228 1994 Thayer GA Yes P11 Cp 19Gresbrecht A 1993 — IL Yes P6 Cp 20 96-2873 1996 Roney AL Yes Did notgrow * Cp 21 URZ298 — — — — P12 Cp 22 FC1 1995 Fitz-Coy East Coast YesP13 Cp 23 Kendall 1993 — IL Yes P4 Cp 24 UDE 95-1372 1995 Fitz-Coy DEYes P14 Cp 25 C97M3 1997 — CO Yes P4 Cp 26 Reed 1993 — IL Yes P4 Cp 27AU2 1996 Roney AL Yes Gangrenous P7 Dermatitis Cp 28 A1A 2002 Skinner DEYes P15 Cp 29 96-7414 1996 Roney AL Yes P13 Cp 30 94-5230 1994 Thayer GAYes P6 Cp 31 94-5224 1994 Thayer GA Yes P6 Cp 32 FC2 1995 Fitz-Coy EastCoast Yes P4 Cp 33 94-5229 1994 Thayer GA Yes P6 Cp 34 7998C 1995 —Canada Yes P1 Cp 35 S1-1 2000 Fitz-Coy East Coast Yes P6 Cp 36 94-52271994 Thayer GA Yes P6 Cp 37 Jones 1993 — IL Yes P6 Cp 38 6A 2002 SkinnerNJ Yes P14 Cp 39 S1-7 2000 Fitz-Coy East Coast Yes NT Cp 40 7998A 1995Roney Canada Yes P1 Cp 41 95-1000 1995 Fitz-Coy — — P4 Cp 42 AU3 1996Roney — — NT * Isolate failed to grow. NT = Not Typed.All isolates were from intestines unless otherwise noted. All isolateswere of chicken origin.

A dendrogram portraying the genetic diversity of various C. perfringensstrains based on SmaI-digested PFGE patterns of C. perfringens DNA isshown in FIG. 2. Among the strains making up the 15 PFGE types, 16strains (about 46%) were grouped in PFGE types P6 (10 strains) and P4 (6strains). The remaining 20 strains clustered into eight PFGE typesrepresented by a single strain (PFGE types P2, P5, P8, P9, P10, P11, P12and P15), four PFGE types represented by two strains each (PFGE typesP3, P7, P13 and P14), and one PFGE type represented by three strains(PFGE type P1). While some of the strains within the same PFGE type wereassociated with the same geographic location/source/year of isolation(e.g., both strains in PFGE type P3 have come from the Illinois DiseaseLab, and they both were isolated in 1995), the number of strains in thePFGE types other than P4 and P6 was too small for making generalizedconclusions about their specific association with any givenfacility/location. Strains in the PFGE types P4 and P6 did not appear tobe associated with a specific geographic location/source of isolation(e.g., strains in the PFGE type P6 were isolated from various sources inIllinois, Georgia and California). FIG. 3 shows phage plaques producedby representative bacteriophage infecting C. perfringens strain Cp 42.

In sum, four to six candidate bacteriophages lytic for C. perfringenswere isolated on phylogenetically distinct strains from environmentalwater sources each obtained from a different poultry farm or processingplant.

Example 2 Characterization of Phages Capable of Infecting Clostridiumperfringens

The methods from Example 1 were also used in Example 2 whereappropriate.

Phage Sterility:

Microbial contamination was determined by (1) plating 1 mL aliquots oftest sample on LB agar plates and incubating replicate plates at 37° C.and 30° C. for 48 hours and (2) pre-incubating 1 mL aliquots of testsample at 37° C. for 24 hours then plating the samples on LB agar andincubating the plates for 24 hours at 37° C. One set of plates wasincubated aerobically and another set anaerobically as indicated. Anybacterial growth at the indicated times denotes contamination.

Phage Purity:

Purity of phage stocks was determined by pulsed-field gelelectrophoresis (PFGE) of uncut DNA. Approximately 100-200 ng of thephage DNA was electrophoresed in a 1% SeaKem® Gold Agarose (Cambrex,Rockland, Me.) gel with 0.5× Sodium boric acid (1×SB: 10 mM sodiumhydroxide pH adjusted to 8.5 with boric acid) buffer at 14° C. in a CHEFMapper XA PFGE apparatus (Bio-Rad Laboratories, Hercules, Calif.). Therun time was 12 hours with a voltage of 6 V/cm and a linearly rampedpulse time of 0.06 seconds to 8.53 seconds. The gels were stained withethidium bromide and visualized with UV light.

Nucleic Acid Characterization:

DNA from each batch of bacteriophage Was isolated by a standardphenol-chloroform extraction method. Proteinase K (200 μg/ml) and RNaseA (1 μg/ml) were added to phage samples with a titer ≧1×10⁹ PFU/ml andincubated at 37° C. for 30 minutes followed by 56° C. for an additional30 minutes. SDS/EDTA was add to a final concentration of 0.1% and 5 mMrespectively and incubated at room temperature for 5 minutes. Thesamples were extracted once with buffered phenol, once withphenol-chloroform and once with chloroform. Phage DNA was ethanolprecipitated and resuspended in 10 mM Tris-HCl (pH 8.0)-0.1 mM EDTA (TE)buffer.

Restriction maps of the phage genomes were made by digestingapproximately 1 μg of the phage DNA with 10 units of XmnI (New EnglandBiolabs, Beverly, Mass.) according to the manufacturers'recommendations. Restriction fragments were separated on a 1.0% agarosegel for 16 hours at 20 V in 1× Tris-acetate-EDTA (10×TAE, EMD Chemicals)buffer and bands visualized by staining with ethidium bromide.

Protein Characterization:

Phage proteins were analyzed by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE). Briefly, phage samples with a titer≧1×10⁸ PFU/ml were denatured in a boiling water bath for 5 minutes inNuPAGE® LDS buffer fortified with DTT (Invitrogen, Carlsbad, Calif.).Aliquots were electrophoresed in a precast NuPAGE® Novex 4 to 12%Bis-Tris continuous gradient gel (Invitrogen) at 120 V for 110 minutes.Proteins was visualized on gels by silver-staining using a SilverXPress®(Invitrogen) according to the manufacturers' recommendations.

Clostridium perfringens Phage Susceptibility:

Forty-six C. perfringens strains were screened for their susceptibilityto the six monophages and two cocktails by the drop on lawn method.Strains was streaked onto BHI agar plates and incubated at 37° C.anaerobically overnight. One colony of each strain was inoculated into aseparate 15-ml culture tube containing BHI broth and incubated at 37° C.anaerobically until the OD₆₀₀ reached 0.1-0.3. One hundred microlitersof each strain was mixed with BHI soft-agar and poured onto a BHI agarplate. After the soft-agar hardened 10 μL of each phage was spotted intriplicate onto the plates inoculated with the C. perfringens strains.Lytic activity was observed after overnight anaerobic incubation at 37°C.

Preparation of Phage Manufacturing Batches:

Shake flask batches of each phage were carried out in 2-L flaskscontaining 1.5 L of BHI broth. Clostridium perfringens strains weregrown in BHI broth anaerobically overnight at 37° C., subcultured andgrown to an OD₆₀₀ of 0.1-0.3. Cultures were infected at an MOIpreviously determined to be optimal for each phage (See Table 5.1.4).Growth was monitored spectrophotometrically until lysis occurred andphage harvested by vacuum filtration (Stericup, Millipore). Batches ofeach phage were concentrated separately and buffer exchanged with PBS bytangential flow filtration in a Pellicon® 2 Mini Cassette using a 50 kDafilter (Millipore).

Endotoxin Levels:

Endotoxin levels for all lots of phage produced were determined by aLimulus amoebocyte lysate (LAL) assay using a chromogenic assay(QCL-1000 Kit, BioWhittaker, Walkersville, Md.) following themanufacturers' recommendations. A standard curve with E. coli endotoxin(supplied with kit) ranging from 0.1 to 1.0 endotoxin units (EU) permilliliter endotoxin was constructed for each assay by plotting theoptical density at 405 nm (OD₄₀₅) versus EU/ml. Phage samples wereassayed diluted in sterile water (USP, Baxter, Deerfield, Ill.) and theabsorbance at 405 nm was measured with a μQuant (Bio-Tek Instruments,Inc., Winooski, Vt.) microplate reader.

Endotoxin concentrations for each sample were calculated by linearregression from the standard curve. Standards and samples were analyzedin triplicate.

Carbohydrate Content:

Carbohydrate content for all lots of phage produced was determined by ananthrone method. A standard curve with glucose ranging from 10 to 250μg/mL was constructed for each assay by plotting the optical density at625 nm (OD₆₂₅) versus μg/mL of glucose concentration. Phage samples wereassayed in PBS and the absorbance at 625 nm was measured with a μQuant™(Bio-Tek Instruments, Inc.) microplate reader.

Carbohydrate concentrations for each sample were calculated by linearregression from the standard curve. Standards and samples were analyzedin duplicate.

Total Protein Content:

The total protein content for all lots of phage produced was determinedby a bicinchoninic acid (BCA) assay using a colorimetric assay (BCA™Protein Assay Kit, Pierce, Rockford, Ill.) following the manufacturer'srecommendations. A standard curve with bovine serum albumin (BSA,supplied with kit) ranging from 20 to 2,000 μg/mL protein wasconstructed for each assay by plotting the optical density at 562 nm(OD₅₆₂) versus μg/mL BSA. The absorbance at 562 nm was measured with aμQuant® (Bio-Tek Instruments, Inc.) microplate reader.

Total protein concentrations for each sample were calculated by linearregression from the standard curve. Standards and samples were analyzedin triplicate.

Electron Microscopy:

High-titer phage lysates (10⁸ PFU/ml) were centrifuged 31,000×g for 2hours and resuspended in 100 mM ammonium acetate (pH 7.0). A drop ofphage suspension was deposited on a carbon-coated Formvar copper grid of400 mesh. The phages were negatively stained by adding a drop ofpotassium phosphotungstate (1-2%, pH 7) and after one minute the excessfluid was withdrawn. Pictures of the phage particles were taken with aPhilips EM 300 transmission electron microscope at an accelerationvoltage of 60 kV with a primary magnification of 29,700×.

Results:

Six novel bacteriophages that infect Clostridium perfringens wereisolated from environmental water or sewage sources. Each phage wascharacterized at both the protein and nucleic acid level. The optimalhost strain for propagation of each bacteriophage was identified and allwere negative for endogenous phage. (Table 2)

TABLE 2 Optimal C. perfringens host strains for propagating C.perfringens-specific phages Phage Host CPLV-42 Cp 27 CPAS-7 Cp 8 CPAS-12Cp 26 CPTA-37 Cp 27 CPAS-15 Cp 8 CPAS-16 Cp 42

Stocks of all six monophages and their respective host strains were madefor use in characterization and production of each phage.

PFGE analysis of uncut DNA from each of the isolated phages showed thatthey were pure monophages with genome sizes of 36 to 50 kb (FIG. 4). DNAfrom each of the monophages isolated was digestible with XmnI (FIG. 5).All of the monophages showed different protein profiles on SDS-PAGE orRFLP profiles confirming that all six monophages are different from oneanother. (FIG. 6)

Electron microscopy showed the bacteriophages to be members of theSiphoviridae or Myoviridae families of icosahedral head phages (FIG. 7)with long tails and double-stranded DNA genomes.

The ability of the six C. perfringens-specific monophages to infect 46C. perfringens strains is shown in Table 3. Phage CPLV-42, CPAS-16,CPTA-37, CPAS-7, CPAS-12, and CPAS-15 infected 20%, 87%, 13%, 39%, 74%,and 52% of the strains screened respectively. The specificity of thephages for C. perfringens was examined by screening the susceptibilityof ten strains of E. coli, L. monocytogenes, S. enterica, and P.aeruginosa. None of these 40 strains were infected by any of themonophages isolated against C. perfringens (Table 4).

TABLE 3 Susceptibility of C. perfringens strains to C.perfringens-specific bacteriophages Phage Cocktail Strain CPLV-42CPAS-16 CPAS-12 CPAS-15 CPAS-7 CPTA-37 INT-401 INT-402 ATCC13124 + + + + + − + + Cp 1 − + − − − − + + Cp 2 − − + + + − + + Cp 3− + + − − − + + Cp 4 − + + + − − + + Cp 5 − + + + − − + + Cp 6 − + + − −− + + Cp 7 + + + + + − + + Cp 8 + + − + + − + + Cp 9 − + + + − − + + Cp10 − + − + − − + + Cp 11 − + + − − − + + Cp 12 − + + − − − + + Cp 13− + + + − − + + Cp 14 − + − + + − + + Cp 15 + + + − − − + + Cp 16− + + + + − + + Cp 17 − + + + + − + + Cp 19 − + + − − − + + Cp 21 − + +− − − + + Cp 22 + + + + − − + + Cp 23 − + + + + − + + Cp 24 − + + + −− + + Cp 25 − + + + + − + + Cp 26 − + + − − − + + Cp 27 + + + − − + + +Cp 28 − + − − − − + − Cp 29 − + − − + − + − Cp 30 − + + − − − + + Cp 31− − + − − − + − Cp 32 + + − + + + + − Cp 33 − + + + − − + − Cp 34 − −− + + + + + Cp 35 − + − + + − + + Cp 36 − − + + − − + + Cp 37 − + + − −− + + Cp 38 − + + + + − + + Cp 39 − + − − − − + + Cp 40 − + − − − − + +Cp 41 − − + − + + + + Cp 42 + + + + + + + + Cp 43 − + + − − − + + Cp 44− + + − + − + + Cp 45 − + + − − − + + Cp 46 − − − − − − − − Cp47 + + + + + + + +

TABLE 4 Susceptibility of other bacterial strains to C.perfringens-specific bacteriophages Phage CPLV- CPAS- CPAS- CPAS- CPAS-CPTA- Strain 42 16 12 15 7 37 Pseudomonas aeruginosa Pa 1 − − − − − − Pa3 − − − − − − Pa 7 − − − − − − Pa 15 − − − − − − Pa 21 − − − − − − Pa 33− − − − − − Pa 42 − − − − − − Pa 62 − − − − − − Pa 65 − − − − − − Pa 72− − − − − − Salmonella enterica SE 24 − − − − − − SS 28 − − − − − − ST31 − − − − − − SHE 43 − − − − − − SH 49 − − − − − − S 45 − − − − − − SAE 72 − − − − − − SK 103 − − − − − − SR 114 − − − − − − SH 162 − − − − −− Listeria monocytogenes Lm 6 − − − − − − Lm 10 − − − − − − Lm 23 − − −− − − Lm 31 − − − − − − Lm 35 − − − − − − Lm 49 − − − − − − Lm 62 − − −− − − Lm 67 − − − − − − Lm 79 − − − − − − Lm 86 − − − − − − Escherichiacoli Ec 3 − − − − − − Ec 26 − − − − − − Ec 37 − − − − − − Ec 41 − − − −− − Ec 56 − − − − − − Ec 60 − − − − − − Ec 65 − − − − − − Ec 68 − − − −− − Ec 73 − − − − − − Ec 77 − − − − − −

The following strains were used to demonstrate the overall activity ofthe phage cocktail INT-401, versus a standardized set of Clostridiumperfringens strains available from depositories. Strains were evaluatedfor lysis by spotting 10 microliters containing 10⁸ pfu/ml onto purelawns of each test strain spread onto BHI agar, and incubating overnightat 37° C.

TABLE 5 Depository Strain Identifier Number INT-401 Lysis ATCC 25768 +ATCC 3624 + ATCC 9856 + ATCC 3628 + ATCC 13124 + ATCC PTA-8495 + NRRLB-50143 (CP8) + NRRL B-50144 (CP26) + NRRL B-50145 (CP42) +

6 monophages were then isolated. (Table 6) Two phage cocktails, INT-401(CPAS-7, CPAS-12, CPAS-15, CPAS-16, and CPLV-42) and INT-402 (CPAS-12,CPAS-15, CPAS-16, CPLV-42), were prepared from five of the sixmonophages isolated. Table 7 gives the levels of endotoxin, totalcarbohydrate, and total protein in C. perfringens-specific cocktails.Both cocktails were effective in killing greater than 85% of the 46 C.perfringens strains screened. INT-401 was selected for use inproof-of-principle efficacy studies designed to determine the preventionof necrotic enteritis in C. perfringens challenged broiler chickens.This cocktail contains the five bacteriophages with the broadest hostrange and is likely to provide a broader (compared to INT-402) spectrumof activity in actual use against wild-type C. perfringens strains

TABLE 6 Summary of C. perfringens monophage batch preparation HostCulture Time Titer Phage Strain MOI (h) (PFU/ml) CPLV-42 Cp 27 1 5  5 ×10¹⁰ CPAS-16 Cp 42 1 3 1 × 10⁸ CPAS-12 Cp 26 1 4-5 1 × 10⁸ CPTA-37 Cp 271 4.5 4 × 10⁸ CPAS-7 Cp 8 1 4-5 5 × 10⁸ CPAS-15 Cp 8 1 4-5 6 × 10⁹

TABLE 7 Levels of endotoxin, total carbohydrate, and total protein in C.perfringens-specific cocktails Content INT-401 INT-402 Endotoxin (EU/ml)384 288 Total Carbohydrate 170 43 (μg/ml) Total protein (μg/ml) 661 175

Example 3 Protocol for Bacteriophage Treatment as Therapy or Preventionof Necrotic Enteritis in Broiler Chickens Challenged with ClostridiumPerfringens

Broiler Chickens:

A total of 576 male day-old broiler chickens were assigned to treatmenton day 0. There were no vaccinations (Mareks or Bronchitis) orantibiotics applied to eggs or chicks at the hatchery.

Housing:

The 64-pen broiler chicken research facility at Maple Leaf Agresearchwas used to conduct the study. Forty-eight pens, each providingapproximately 10 square feet of floor space, were assigned to treatmentgroups. Each pen had a concrete floor and nylon mesh partitionssupported by PVC frame. Adjacent pens were separated by a solid 12-inchhigh plastic bather at bird level. Each pen was permanently identifiedby number and contained 12 birds on day zero. The barn was heated by twonatural gas heaters, which were equally spaced and positioned to warmincoming air at the south wall of the building. Air was exhausted byfans located on the north-facing wall of the building. Each pencontained one nipple-type drinker, which provided clean drinking waterad libitum. Water was de-chlorinated. Dry feed was provided ad libitumin trough-type feeders (one per pen) of 5-kg capacity. New wood shavingswere used as bedding.

Management:

Lighting program, barn temperature, litter type and other managementpractices were typical of commercial broiler chicken producers in thelocal geographic area and is fully documented in the raw data. Birds,which were moribund and unable to reach food or water, were culled andeuthanized by carbon dioxide gas.

Bodyweight, pen number, date of death and cause of death were determinedby necropsy and recorded for each bird culled or found dead during thestudy.

Experimental Design:

A randomized complete block design was used to study the effects ofeight treatments. The treatments were as follows:

TABLE 8 Treatment design Treatment C. perfringens code Challenge TestArticle 1 No No 2 Yes No 3 Yes Yes 4 Yes Yes 5 Yes Yes 6 Yes Yes 7 YesYes 8 Yes Yes

There were 8 pens per block (8 treatments) and 6 blocks (replicates) fora total of 48 pens. (See Section 4.2, Deviation #2 for change to abovetreatment to block assignment).

Treatment Groups:

Treatment 1—Control—UUC (no C. perfringens challenge or bacteriophageadministration)

Treatment 2—Control—IUC (C. perfringens challenge without bacteriophagecocktail)

Treatment 3—In ovo injection of phage cocktail at day 18 of incubation.

Treatment 4—Spray application of phage cocktail to chicks afterhatching.

Treatment 5—In ovo injection of phage cocktail at day 18 of incubationand spray application of phage cocktail to chicks after hatching.

Treatment 6—In ovo injection of phage cocktail at day 18 of incubation,spray application of phage cocktail to chicks after hatching and oralgavage of bacteriophage cocktail on Day 7 through 13.

Treatment 7—Bacteriophage cocktail administered via oral gavage from Day7 through Day 13 (oral gavage ceased on day of Clostridium perfringenschallenge)

Treatment 8—Bacteriophage cocktail administered via oral gavagebeginning on Day 14 (concurrent with Clostridium perfringens challenge)through Day 18.

Feeding Program:

The following feeding program was used in the study:

TABLE 9 Feeding program Formulation Day Feed Type number*  0-13 Starter282 9:00 p.m. Day13 to None. Feed was None 9:00 a.m. Day 14 withdrawn14-21 Starter 282

Feed Sampling:

The investigator's representative was present during feed manufacture.Ten representative samples were taken from each batch of final feed,composited and divided into three samples for proximate analysis, andretainer samples, respectively.

Administration of Clostridium perfringens Challenge:

A Clostridium perfringens isolate originating from a field case ofnecrotic enteritis in Ontario was used in the study. Inoculum containedapproximately 10⁸ cfu Clostridium perfringens per mL at time of feeding.Feed was withdrawn from all birds for approximately 8 hours prior tofirst introduction of challenge. Inoculum was administered to birds viafeed in the afternoon and night commencing Day 14 P.M. and ending Day 15A.M. using trough-type feeders. A suitable quantity of assigned feed(approximately 0.150 kg) and an amount of inoculum equal toapproximately 1.5 times the weight of feed was added to each feeder.When this procedure was complete for all pens assigned to challenge,feeders were returned to their corresponding pens. Inoculum-feed mixtureremaining at the end of the half-day period was weighed and discarded.

Lesion Scoring of Sacrificed Birds:

Three birds were randomly selected from each pen on Day 16 andeuthanized. These birds were scored grossly for necrotic enteritis andcoccidiosis lesions:

TABLE 10 Necrotic enteritis scoring Necrotic enteritis score Description0 Normal, no evidence of gross lesions 1 Thin, friable small intestine 2Focal necrosis and/or ulceration 3 Patchy necrosis 4 Severe extensivenecrosis (typically seen in birds which have died from NE)

Clostridium perfringens Culture of Small Intestinal Segment:

A small intestinal segment was collected from 40 birds that died on orafter Day 15 and had a gross diagnosis of necrotic enteritis. Thesegment was forwarded to the Department of Pathology at the Universityof Guelph for C. perfringens culture. Culture results were reported aspositive or negative for C. perfringens. Samples of positive bacterialcultures were forwarded to Intralytix for testing for phagesusceptibility. In addition, 144 ileum samples were collected from thebirds sacrificed for C. perfringens lesion scoring on Day 16. Thesesamples were quantitatively tested for C. perfringens at the abovereferenced laboratory and microbiological samples were forwarded toIntralytix for additional characterization of phage activity.

Necropsy:

All birds that died or were euthanized were submitted to the studypathologist for gross necropsy to determine the cause of death.

Observations and Calculation of Variables:

1) Bodyweight and number of birds per pen on Days 0, 14, and 21.

2) Amounts of each feed consumed by each pen.

3) Bodyweight and date of death for birds which were culled or died.

4) Feed conversion ratio was calculated on a pen basis as feedconsumed/[total weight of live birds+total weight of dead and culledbirds+total weight of sacrificed birds] for the 0-14, 14-21 and 0-21 Dayperiods.

5) Average bodyweight per pen was calculated as total weight of livebirds at time of weighing/number of live birds at time of weighing.

6) Daily feed intake (grams) per live bird day was calculated on a penbasis for Day 0-14, Day 14-21 and Day 0-21.

7) Apparent cause of death was recorded for all birds that died or wereculled. Total mortality and mortality from necrotic enteritis will becalculated on a pen basis.

8) Evaluation of the effects of the in ovo injection treatments onpercent hatch and chick health at the hatchery.

9) Necrotic enteritis lesion score of sacrificed birds (Day 16)

10) Birds were observed on a flock basis at least once daily andobservations recorded.

Test Substance Disposition:

Remaining bacteriophage cocktail test substance was destroyed byincineration and destruction is documented in the study records.

Bird Disposition:

Birds (treated and control) were humanely euthanized at the end of thestudy and disposed of via incineration and method and date ofdisposition was recorded in the study records. Hatchery waste and unusedin ovo bacteriophage injected eggs were disposed of via incineration.Hatched chicks that had been in ovo injected or sprayed withbacteriophage but not assigned to the study, were humanely euthanizedand disposed of via incineration.

Original Data:

Original data is submitted to the sponsor together with the finalreport. An exact copy of the final report and data will be maintained atMaple Leaf Agresearch for a minimum of two years.

Documentation:

All raw (original) data was recorded in black ink on data sheets bearingthe trial number. Corrections were made by drawing a single line throughthe original entry and writing the correct entry beside it together withinitials of the person making the correction, the date the correctionwas made and the reason for the correction. Defined error codes wereused to record reason for correcting a data point.

Statistical Analysis:

Randomized complete block design was be used. Pen location within thefacility was the blocking factor. The pen was the experimental unit forstatistical analysis. A one-way treatment structure was utilized witheach treatment being replicated six times (once within each block,except as detailed in Deviation #2, Section 4.2). Mortality data wastransformed using an arcsine transformation prior to analysis ofvariance. Mixed models analysis was used to analyze all data. Means werecompared using an appropriate multiple range test.

Amendment #1:

This amendment clarified dates, eliminated vaccine administration andany potential interference vaccine might have with the Test Article, anddetailed exact doses of Test article to be administered during “in ovo”injection (0.2 mL), spraying (about 7 mL per 100 chicks) and oral dosing(0.5 mL per bird per day).

Amendment #2:

This amendment redefined the dosages of “in ovo” administration (0.05 mLper egg) and spray (7 to 22 mL per 100 chicks). The upper range of 22 mLwas actually used for the spray.

Deviations:

Two deviations occurred and are described in the Protocol section of theStudy Binder. A brief description follows:

Deviation #1:

Only 12 birds were assigned to pens instead of the 15 described in theprotocol. This will have an impact on the statistical power of thestudy, particularly the mortality data.

Deviation #2:

Block 3 was assigned two treatment 2 pens and no treatment 5 pen causingan imbalance in the design. Least square means will be reported tocorrect for the unequal representation per treatment group. This is notexpected to have a major influence on the power of the study.

Example 4 Results for Bacteriophage Treatment as Therapy or Preventionof Necrotic Enteritis in Broiler Chickens Challenged with Clostridiumperfringens

The results of this study are summarized in Tables 10 to 12. A detailedstatistical analysis was performed.

Hatchery:

“In ovo” injection of eggs with Test Article (0.05 mL per egg) forTreatments 3, 5 and 6 was performed at 18 days of incubation using anEmbrex machine and followed the standard industry protocol with thefollowing exceptions: Marek's vaccine and antibiotic (Excenel) were notincluded. This standard procedure also involved applying a small amountof chlorine solution over the injection hole just post injection. Forthis trial, 1259 fertile eggs were transferred without being injectedand hatched at 96.6%. The 775 fertile eggs injected with Test Articlehatched at 95.5%.

TABLE 11 Delivery Routes of Bacteriophage on weights of broiler chickenschallenged with necrotic enteritis Average live weights (kg) Treatment¹Day 0 Day 14 Day 21 Day 35 Day 42 Control .046 .330 .750^(A) 1.917^(A)2.776^(A) Challenged control .046 .340 .618^(C) 1.530^(C) 2.342^(C) BMDcontrol .045 .340 .634^(C) 1.795^(B) 2.686^(AB) Gavaged phage .045 .328.641^(C) 1.762^(B) 2.601^(B) Phage in water .046 .348 .694^(B)1.812^(AB) 2.664^(AB) Phage in feed .045 .333 .658^(BC) 1.754^(B)2.592^(B) SEM² .000 .010 .018 .045 .059 Pr > F .5309 .7492 .0003 .0001.0004 ¹LSMEANS were provided for each treatment. The treatment groupsincluded a control, challenged control, BMD 50 g/ton as a medicatedcontrol, oral gavaged phage, phage provide via water and phage providevia feed. The bacteriophage used was Intralytix C. perfringens PhageCocktail - 4.8 × 10⁹ pfu/ml. On Day 14, all birds were orally inoculatedwith a coccidial inoculum containing approximately 5,000 oocysts of E.maxima per bird. All groups, except the control, were challenged withClostridium perfringens on Days 18, 19, and 20. Oral Administration ofphage cocktail via gavages, drinking water and feed application willoccurred on days 17, 18, 19, 20, and 21. ²Standard error of the LSMEANS.^(A,B,C,)Means within columns with different superscripts aresignificantly different (P < .05).

Three Treatments (#'s 4, 5 and 6, Table 10) were also sprayed with TestArticle at the hatchery after hatch. A commercial spray cabinet designedfor administering coccidiosis vaccine was used to deliver the TestArticle at a rate of 22 mL per box or approximately 0.22 mL per bird.These birds were held in the hatchery for an extra ½ hour to permitdrying prior to transport to the research farm.

Challenged pens were provided with 1.66 kg of the Clostridia perfringensinoculum/feed mixture and all consumed at least 1.25 kg except one, achallenged control pen. This pen suffered from severe water restrictiondue to a technical problem and for this reason was removed from theanalysis. Due to the deviation described above the challenged control(Treatment 2) was assigned one extra pen and Treatment 5 one less pen.With this slight imbalance in design, least square means are reported.There was no significant (P>0.05) difference between challenged groupsin quantity of inoculum consumed.

The primary criteria for evaluating the effectiveness of Test Articleand its method of administration is mortality attributable directly toClostridia perfringens challenge. No birds died from Necrotic Enteritis(NE) in the non-challenged control (Treatment 1) and this wassignificantly (P<0.01) different than the challenged control with 25.9%percent of birds in a pen dying of NE. Birds treated (Treatment 8) byOral Gavage (OG) from the day of challenge (day 14) until day 18 had thelowest mortality (5.6%) of the phage treated groups and this was notsignificantly (P>0.05) different from the non-challenged control(Treatment 1). Two of the “In ovo” groups (Treatments 3 and 6) hadintermediate NE mortality, which was not significantly (P>0.05)different from either Control groups (Treatments 1 and 2).

Three birds per pen were sacrificed at Day 16 to detect lesions typicalof NE and to determine the presence of Clostridia perfringens in eithera defined segment (approximately 3 to 4 cm distal to the duodenum) if nolesions were present or a segment surrounding an identified lesion.Although not significantly (P>0.05) different from the other treatmentgroups, there were no “typical” NE lesions found in the non-challengedcontrol (Treatment 1). No significant (P>0.05) difference betweentreatments was found for lesion scores.

Clostridia perfringens (Cp) bacterium were isolated from all groupsincluding the non-challenged control. We do not know if the strainisolated from the non-challenged control was the same as the challengedstrain. However, Treatment 1 was numerically lower for Bacterial Scoresfor Cultures and this was consistent with the significantly (P<0.05)lowest score (Tablel) for Smears. No other trends were evident in theBacterial Score means for either Cultures or Smears between the othertreatment groups.

TABLE 12 Delivery Routes of Bacteriophage on weight gains of broilerchickens challenged with necrotic enteritis Average weight gain(kg) DaysDays Treatment¹ 0-14 Days 0-21 14-21 Days 0-35 Days 0-42 Control .284.705^(A) .421^(A) 1.871^(A) 2.730^(A) Challenged control .294 .572^(C).278^(C) 1.484^(C) 2.296^(C) BMD control .295 .589^(C) .294^(C)1.750^(B) 2.641^(AB) Gavaged phage .283 .596^(C) .313^(BC) 1.716^(B)2.556^(B) Phage in water .302 .648^(B) .346^(B) 1.766^(AB) 2.618^(AB)Phage in feed .287 .612^(BC) .325^(BC) 1.709^(B) 2.547^(B) SEM² .010.018 .014 .045 .059 Pr > F .7559 .0003 .0001 .0001 .0004 ¹LSMEANS wereprovided for each treatment. The treatment groups included a control,challenged control, BMD 50 g/ton as a medicated control, oral gavagedphage, phage provide via water and phage provide via feed. Thebacteriophage used was Intralytix C. perfringens Phage Cocktail - 4.8 ×10⁹ pfu/ml. On Day 14, all birds were orally inoculated with a coccidialinoculum containing approximately 5,000 oocysts of E. maxima per bird.All groups, except the control, were challenged with Clostridiumperfringens on Days 18, 19, and 20. Oral Administration of phagecocktail via gavages, drinking water and feed application will occurredon days 17, 18, 19, 20, and 21. ²Standard error of the LSMEANS.^(A,B,C,)Means within columns with different superscripts aresignificantly different (P < 0.05).

There was a significantly (P<0.05) higher chick weight for one of thegroups (Treatment 4) receiving Test Article by Spray at the hatchery.This was not significantly different than one (Treatment 5) of the othertwo groups receiving the Spray. This may be a result of these Treatmentsretaining more moisture from the Spray procedure. Pre-challenge, on Day14, there were no significant differences (P>0.05) in body weightbetween the Treatments. After challenge, at Day 21, the non-challengedcontrol (Treatment 1) was significantly (P<0.05) heavier than the birdsreceiving Test Article by Oral Gavage (Treatment 7) prior to challenge.No other differences in growth performance were detected.

Total mortality in the non-challenged control was high at 13.9%. Asindicated in the necropsy records much of this non-NE mortality was dueto internal infections, omphalitis (yolk sac infections) and suddendeath. This high early chick mortality is not typical. Total mortalitywas significantly (P<0.05) lower for the non-challenged control(Treatment 1, 13.9%) and the birds receiving Oral Gavage from day 14 today 18 (Treatment 8, 12.5%) than Treatment 5 (37.0%).

TABLE 13 Delivery Routes of Bacteriophage on feed conversion of broilerchickens challenged with necrotic enteritis Feed conversion ratio (feedto gain)² Days Days Treatment¹ 0-14 Days 0-21 14-21 Days 0-35 Days 0-42Control 1.703 1.532^(D) 1.417^(C) 1.709^(D) 1.892^(D) Challenged 1.6001.912^(A) 2.284^(A) 2.483^(A) 3.226^(A) control BMD control 1.5611.829^(AB) 2.130^(A) 2.077^(B) 2.652^(B) Gavaged phage 1.662 1.760^(BC)1.864^(B) 1.814^(CD) 2.086^(C) Phage in water 1.562 1.676^(C) 1.778^(B)1.813^(CD) 2.066^(C) Phage in feed 1.680 1.777^(ABC) 1.868^(B) 1.841^(C)2.089^(C) SEM³ .045 .051 .081 .047 .039 Pr > F .1359 .0002 .0001 .0001.0001 ¹LSMEANS were provided for each treatment. The treatment groupsincluded a control, challenged control, BMD 50 g/ton as a medicatedcontrol, oral gavaged phage, phage provide via water and phage providevia feed. The bacteriophage used was Intralytix C. perfringens PhageCocktail - 4.8 × 10⁹ pfu/ml. On Day 14, all birds were orally inoculatedwith a coccidial inoculum containing approximately 5,000 oocysts of E.maxima per bird. All groups, except the control, were challenged withClostridium perfringens on Days 18, 19, and 20. Oral Administration ofphage cocktail via gavages, drinking water and feed application willoccurred on days 17, 18, 19, 20, and 21. ²The feed conversion ratio wasadjusted for the weights of mortality and removed weights. ³Standarderror of the LSMEANS. ^(A,B,C,D,)Means within columns with differentsuperscripts are significantly different (P < 0.05).

TABLE 14 Delivery Routes of Bacteriophage on mortality and lesion scoresof broiler chickens challenged with necrotic enteritis Mortality (%)²Necrotic Totals include all causes Necrotic enteritis Days enteritislesion Treatment¹ Days 0-21 Days 0-35 0-42 Days 0-42 scores³ Control2.67^(CD)  2.67^(D) 4.00^(D)  0^(D) 0^(B) Challenged 41.33^(A) 66.00^(A)66.67^(A) 64.00^(A)  .9^(A) control BMD control 24.67^(B) 51.33^(B)53.33^(B) 50.00^(B) 1.1^(A) Gavaged phage 10.00^(C) 16.67^(C) 18.00^(C)14.00^(C)  .1^(B) Phage in water .67^(D) 67^(D) 3.33^(D)  0^(D)  .1^(B)Phage in feed 2.00^(CD)  3.33^(D) 5.33^(D)  .66^(D)  .4^(B) SEM⁴ 2.97 2.71 2.81  2.76  .2 Pr > F .0001  .0001 .0001  .0001  .0006 ¹LSMEANSwere provided for each treatment. The treatment groups included acontrol, challenged control, BMD 50 g/ton as a medicated control, oralgavaged phage, phage provide via water and phage provide via feed. Thebacteriophage used was Intralytix C. perfringens Phage Cocktail - 4.8 ×10⁹ pfu/ml. On Day 14, all birds were orally inoculated with a coccidialinoculum containing approximately 5,000 oocysts of E. maxima per bird.All groups, except the control, were challenged with Clostridiumperfringens on Days 18, 19, and 20. Oral Administration of phagecocktail via gavages, drinking water and feed application will occurredon days 17, 18, 19, 20, and 21. ²Percentage data were analyzed with andwithout transformation (arc sin square root). ³On Day 22, scoring wasbased on a 0 to 3 score, with 0 being normal and 3 being the mostsevere. ⁴Standard error of the LSMEANS. ^(A,B,C,D,)Means within columnswith different superscripts are significantly different (P < .05).

The non-challenged control (Treatment 1) had the numerically highest(102 grams per bird per day) feed intake and this was significantly(P<0.05) more than Treatment 5 (84 grams per bird per day). Although themeans comparison was not significant (P>0.05), Treatment 8 had thenumerically best FCR (1.477) of the Phage treated groups and equal tothe performance of the non-challenged control.

CONCLUSIONS

1. A successful Clostridia perfringens (Cp) challenge was achieved. Thepositive control had 25.9% of the birds die of Necrotic Enteritiscompared to the negative control (0.0%).

2. Oral Gavage of Test Article to birds on the day of challenge (Day 14)and for the next four days significantly reduced mortality due to NE.Growth performance in this group was numerically equivalent to theNon-Challenged control.

3. Two of the three “In ovo” treatments had numerically reduced NEmortality (9.6 and 14.8%) when compared to the Challenged control(25.9%).

4. Oral Gavage of Test Article prior to challenge was ineffective inpreventing NE mortality due to Cp challenge.

5. Spray of Test Article to chicks at the hatchery did not significantly(P>0.05) reduce NE mortality from Cp challenge.

6. The precision of this trial was reduced by several factors includingfewer birds being assigned to pens at day old than specified in theprotocol and high early non-challenge related mortality.

Example 5 Sequence Analysis of C. perfringens Bacteriophage

Media:

Brain Heart Infusion (BHI) broth or BHI agar supplemented with 250 mg/Lcycloserine was used to grow all C. perfringens isolates. Luria-Bertani(LB) broth and LB agar was used to grow all aerobic strains. All mediawere obtained from EMD Chemicals, Gibbstown, N.J.

Microorganisms:

Clostridium perfringens strains were from the Intralytix, Inc. CultureCollection, Baltimore, Md. As part of the collection process, isolateswere checked for purity and frozen at −80° C. in 30% glycerol. Most ofthe work was performed in an anaerobic chamber (Plan-labs, Lansing,Mich.), that contained a 90% N₂-5% 11₂-5% CO₂ atmosphere. Escherichiacoli, Listeria monocytogenes, Salmonella enterica, and Pseudomonasaeruginosa strains were from the Intralytix, Inc. Culture Collection andall were grown aerobically.

Bacteriophage:

All bacteriophages were isolated from environmental water, industrialwastewater, or sewage sources.

Phage DNA Isolation:

DNA from each batch of bacteriophage was isolated by a standardphenol-chloroform extraction method. Proteinase K (200 μg/mL) and RNaseA (1 μg/mL) were added to phage samples with a titer Z1×10⁹ PFU/ml andincubated at 37° C. for 30 minutes followed by 56° C. for an additional30 minutes. SDS/EDTA was add to a final concentration of 0.1% and 5 mMrespectively and incubated at room temperature for 5 minutes. Thesamples were extracted once with buffered phenol, once withphenol-chloroform and once with chloroform. Phage DNA was ethanolprecipitated and resuspended in 10 mM Tris-HCl (pH 8.0)-0.1 mM EDTA (TE)buffer.

Phage Sequencing:

The DNA from each of the phages was sequenced using standard automatedsequencing methods.

Sequence Analysis:

To identify the predicted open reading frames (ORFs) WPA uses acombination of CRITICA (1) and GLIMMER (2). The results from theseprograms are combined and the optimal open reading frames are extractedfrom the combined data set.

Each of the two programs uses different algorithms for identifying openreading frames, and each has its benefits and drawbacks. However, bycombining the output from both tools WPA is able to optimize thepredicted ORFs that they can identify.

WPA uses an automated annotation system in which assignments aregenerated primarily by sequence similarity searches between the de novoidentified ORFs and other ORFs in their databases. In this case, theBLAST algorithm was used to compare predicted protein sequences for theannotation. In addition to the publicly available databases forcomparison WPA has a phage database that contains approximately 400phage genomes which they feel represents the best phage datasetavailable.

Following the automated annotation and assignment phase, the assignmentsfor each gene are manually curated by Intralytix to see if any of the 17undesirable genes (Table 15) are present.

TABLE 15 List of undesirable genes encoded in bacteriophage genomesToxin and its Encoding Gene Bacterial Pathogen Enterotoxin A (entA)Staphylococcus aureus Enterotoxin A (sea, sel) StaphylococcusEnterotoxin A (sea) Staphylococcus aureus Staphylokinase (sak)Staphylococcus aureus Enterotoxin P (sep) Staphylococcus aureusExfoliative toxin A (eta) Staphylococcus aureus Diphtheria toxin (tox)Corynebacterium diphtheriae Shiga toxins (stx1,2) Escherichia coliCytotoxin (ctx) Pseudomonas aeruginosa Cholera toxin (ctxA) Vibriocholerae Cholera toxin (ctxB) Vibrio cholerae Zonula occludens toxin(zot) Vibrio cholerae Neurotoxin (C1) Clostridium botulinumEnterohaemolysin (hly) Escherichia coli Streptococcal exotoxin AStreptococcus pyogenes (speA) Streptococcal exotoxin C Streptococcuspyogenes (speC) Streptococcal exotoxin K Streptococcus pyogenes (speK)

Five of the six phages were sequenced. The sequences of each of the fivephage genomes were obtained and each predicted open reading frameidentified in each genome (Table 16). Each of the predicted genes wasannotated. None of the 17 undesirable genes (Table 15) were found in thegenomes of any of the five phages for which sequences were available(Table 17).

TABLE 16 Number of predicted ORFs for each C. perfringens-specificbacteriophage Number of Open Reading Frames Phage (ORFs) 1 67 2 77 3 694 47 5 67

TABLE 17 Annotations of all predicted genes for each C.perfringens-specific bacteriophage genome Gene ID Annotated FunctionCPAS-7 1 Presumed portal vertex protein 2 Ring-infected erythrocytesurface antigen 3 Hypothetical protein 4 Hypothetical protein 5FKBP-type peptidyl-prolyl cis-trans isomerase (trigger factor) 6Isocitrate dehydrogenase kinase/phosphatase 7 Phage-like element PBSXprotein xkdH 8 Phase 1 flagellin 9 Type I restriction-modificationsystem restriction subunit (EC 3.1.21.3) 10 Sarcosine oxidase, alphasubunit 11 Hypothetical protein 12 Phage-like element PBSX protein xkdK13 Phage-like element PBSX protein xkdM 14 Hypothetical protein 15Hypothetical protein 16 Phage protein 17 Hypothetical protein 18Phage-like element PBSX protein xkdQ 19 Ribosomal protein S4 and relatedproteins 20 Phage-like element PBSX protein xkdS 21 Hypothetical protein22 Phage-like element PBSX protein xkdT 23 Tail fiber 24 Heat shockprotein 90 25 Hypothetical protein 26 Bacteriocin uviB precursor 27N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) 28 ABC transporter,permease protein 29 No hits 30 Transposase 31 DNA repair protein RadA 32Transcriptional regulator 33 Hypothetical protein 34 Hypotheticalprotein 35 Thymidine kinase (EC 2.7.1.21) 36 Tryptophanyl-tRNAsynthetase (EC 6.1.1.2) 37 CMP-binding factor 38 3-isopropylmalatedehydratase large subunit (EC 4.2.1.33) 39 DNA ligase 40 Putativepenicillin-binding protein 41 Signal transducer and activator oftranscription 1 42 Chemotaxis protein CHED 43 Gramicidin S synthetase I(EC 5.1.1.11) 44 ABC transporter ATP-binding protein 45 PutativeATP-dependent DNA helicase 46 DNA polymerase I 47 Hypothetical protein48 Hypothetical protein 49 Phenylalanyl-tRNA synthetase beta chain 50Terminase large subunit 51 Terminase small subunit 52 Hypotheticalprotein 53 CobT protein 54 Putative chromosome segregation protein, SMCATPase superfamily 55 Deoxycytidylate deaminase (EC 3.5.4.12) 56 aceE;pyruvate dehydrogenase e1 component oxidoreductase protein 57Hypothetical protein 58 Hypothetical protein 59 Aspartic acid-richprotein aspolin2 60 CDEP 61 Nucleolin 62 Peptide ABC transporter,ATP-binding protein 63 GTP-binding protein SAR1 64 gp56 dCTPase 65Hypothetical protein 66 Homeobox-leucine zipper protein 67 DNA repairprotein recN CPAS-16 1 Developmentally regulated GTP-binding protein 1 2Presumed portal vertex protein 3 Ring-infected erythrocyte surfaceantigen 4 Hypothetical protein 5 Hypothetical protein 6 FKBP-typepeptidyl-prolyl cis-trans isomerase (trigger factor) 7 Isocitratedehydrogenase kinase/phosphatase 8 Phage-like element PBSX protein xkdH9 High-affinity potassium transporter 10 Sarcosine oxidase, alphasubunit 11 Hypothetical protein 12 Phage-like element PBSX protein xkdK13 Phage-like element PBSX protein xkdM 14 Hypothetical protein 15Hypothetical protein 16 Phage protein 17 Hypothetical protein 18Phage-like element PBSX protein xkdQ 19 Ribosomal protein S4 and relatedproteins 20 Phage-like element PBSX protein xkdS 21 Hypothetical protein22 Phage-like element PBSX protein xkdT 23 Tail fiber 24 Heat shockprotein 90 25 Hypothetical protein 26 Bacteriocin uviB precursor 27N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) 28 Enterotoxin 29Membrane protein 30 No hits 31 Thymidylate synthase (EC 2.1.1.45) 32Heat shock protein (dnaJ-2) 33 Hypothetical protein 34 DNA polymerase I35 DNA polymerase I 36 Putative ATP-dependent DNA helicase 37 ABCtransporter ATP-binding protein 38 Terminase large subunit 39 Terminasesmall subunit 40 CobT protein 41 Putative chromosome segregationprotein, SMC ATPase superfamily 42 Deoxycytidylate deaminase (EC3.5.4.12) 43 aceE; pyruvate dehydrogenase e1 component oxidoreductaseprotein 44 Hypothetical protein 45 Hypothetical protein 46 Genomic DNA,chromosome 3, BAC clone: F1D9 47 SWF/SNF family helicase 48 Asparticacid-rich protein aspolin2 49 CDEP 50 Nucleolin 51 Hypothetical protein52 Putative reductase 53 Hypothetical protein 54 Hypothetical protein 55Hypothetical protein 56 Tail fiber 57 gp56 dCTPase 58 Acetate kinase (EC2.7.2.1) 59 GTP-binding protein SAR1 60 Peptide ABC transporter,ATP-binding protein 61 Cytochrome b (EC 1.10.2.2) 62 Homeobox-leucinezipper protein 63 DNA repair protein recN 64 Transposase 65 DNA repairprotein RadA 66 Exonuclease I 67 Transcriptional regulator 68Hypothetical protein 69 Thymidine kinase (EC 2.7.1.21) 70Tryptophanyl-tRNA synthetase (EC 6.1.1.2) 71 CMP-binding factor 723-isopropylmalate dehydratase large subunit (EC 4.2.1.33) 73 DNA ligase74 Putative penicillin-binding protein 75 Signal transducer andactivator of transcription 1 76 Chemotaxis protein CHED 77 Gramicidin Ssynthetase I (EC 5.1.1.11) CPAS-15 1 Gramicidin S synthetase I (EC5.1.1.11) 2 Chemotaxis protein CHED 3 Signal transducer and activator oftranscription 1 4 Putative penicillin-binding protein 5 DNA ligase 63-isopropylmalate dehydratase large subunit (EC 4.2.1.33) 7 CMP-bindingfactor 8 Tryptophanyl-tRNA synthetase (EC 6.1.1.2) 9 Thymidine kinase(EC 2.7.1.21) 10 Hypothetical protein 11 Hypothetical protein 12Transcriptional regulator 13 Exonuclease I 14 DNA repair protein RadA 15Transposase 16 ABC transporter ATP-binding protein 17 Y56A3A.29a protein18 DNA polymerase I 19 DNA polymerase I 20 Thymidylate synthase (EC2.1.1.45) 21 Hypothetical protein 22 SWF/SNF family helicase 23 Asparticacid-rich protein aspolin2 24 CDEP 25 Hypothetical protein 26 Terminaselarge subunit 27 Terminase small subunit 28 CobT protein 29 Putativechromosome segregation protein, SMC ATPase superfamily 30Deoxycytidylate deaminase (EC 3.5.4.12) 31 aceE; pyruvate dehydrogenasee1 component oxidoreductase protein 32 Hypothetical protein 33Hypothetical protein 34 DNA repair protein recN 35 Homeobox-leucinezipper protein 36 Hypothetical protein 37 No hits 38 Hypotheticalprotein 39 N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) 40Bacteriocin uviB precursor 41 Hypothetical protein 42 Heat shock protein90 43 Tail fiber 44 PROBABLE SUCCINYL-COA SYNTHETASE BETA CHAIN PROTEIN45 Phage-like element PBSX protein xkdT 46 Hypothetical protein 47Phage-like element PBSX protein xkdS 48 Ribosomal protein S4 and relatedproteins 49 Phage-like element PBSX protein xkdQ 50 Hypothetical protein51 Phage protein 52 Phage protein 53 Hypothetical protein 54Hypothetical protein 55 Phage-like element PBSX protein xkdM 56Phage-like element PBSX protein xkdK 57 Hypothetical protein 58Sarcosine oxidase, alpha subunit 59 High-affinity potassium transporter60 Phage-like element PBSX protein xkdH 61 Putative nodulation protein62 Isocitrate dehydrogenase kinase/phosphatase 63 FKBP-typepeptidyl-prolyl cis-trans isomerase (trigger factor) 64 Hypotheticalprotein 65 Hypothetical protein 66 Ring-infected erythrocyte surfaceantigen 67 Presumed portal vertex protein 68 GTP-binding protein SAR1 69Hypothetical protein CPLV-42 1 Hypothetical protein 2 Ribosomal proteinS4 and related proteins 3 Hypothetical protein 4 F14H3.11 protein 5Myosin heavy chain, cardiac muscle beta isoform 6 Holin 7N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) 8 Developmentallyregulated GTP-binding protein 1 9 Hypothetical protein 10 DNAinternalization-related competence protein ComEC/Rec2 11 Phage-relatedprotein 12 Antirepressor 13 Phage protein 14 Hypothetical protein 15Excinuclease ABC subunit B 16 Putative oxidoreductase protein 17 DNAtopoisomerase I (EC 5.99.1.2) 18 Imidazole glycerol phosphate synthasesubunit hisF (EC 4.1.3.—) 19 Methyl-accepting chemotaxis protein 20Hypothetical protein 21 Aminomethyltransferase (EC 2.1.2.10) 22Hypothetical protein 23 Hypothetical protein 24 Hypothetical protein 25Hypothetical protein 26 Hypothetical protein 27 Hypothetical protein 28DNA polymerase III, subunit beta 29 Terminase large subunit 30 lin258531 Portal protein 32 Genomic DNA, chromosome 3, BAC clone: F1D9 33Deoxyguanosinetriphosphate triphosphohydrolase (dgtP) 34Virulence-associated protein E 35 Hypothetical protein 363′-phosphoadenosine 5′-phosphosulfate sulfotransferase (PAPSreductase)/FAD synthetase and related enzymes, COG0175 37Phosphoadenosine phosphosulfate reductase (EC 1.8.4.8) 38 Hypotheticalprotein 39 Hypothetical protein 40 WRKY transcription factor 22 41D-threonine dehydrogenase 42 Hypothetical protein 43 Hypotheticalprotein 44 Transcriptional regulator 45 Single-strand binding protein 46Hypothetical protein 47 Sensor histidine kinase CPAS-12 1 Presumedportal vertex protein 2 Ring-infected erythrocyte surface antigen 3Hypothetical protein 4 Hypothetical protein 5 FKBP-type peptidyl-prolylcis-trans isomerase (trigger factor) 6 Isocitrate dehydrogenasekinase/phosphatase 7 Phage-like element PBSX protein xkdH 8High-affinity potassium transporter 9 Sarcosine oxidase, alpha subunit10 Hypothetical protein 11 Phage-like element PBSX protein xkdK 12Phage-like element PBSX protein xkdM 13 Hypothetical protein 14Hypothetical protein 15 Phage protein 16 Hypothetical protein 17Phage-like element PBSX protein xkdQ 18 Ribosomal protein S4 and relatedproteins 19 Phage-like element PBSX protein xkdS 20 Hypothetical protein21 Phage-like element PBSX protein xkdT 22 Tail fiber 23 Heat shockprotein 90 24 Hypothetical protein 25 Bacteriocin uviB precursor 26N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) 27 ABC transporter,permease protein 28 No hits 29 Gramicidin S synthetase I (EC 5.1.1.11)30 Chemotaxis protein CHED 31 Signal transducer and activator oftranscription 1 32 Putative penicillin-binding protein 33 DNA ligase 343-isopropylmalate dehydratase large subunit (EC 4.2.1.33) 35 CMP-bindingfactor 36 Tryptophanyl-tRNA synthetase (EC 6.1.1.2) 37 Thymidine kinase(EC 2.7.1.21) 38 Nucleolin 39 Transcriptional regulator 40 DNA repairprotein RadA 41 Transposase 42 Hypothetical protein 43 Thymidylatesynthase (EC 2.1.1.45) 44 Heat shock protein (dnaJ-2) 45 DNA polymeraseI 46 Putative ATP-dependent DNA helicase 47 ABC transporter ATP-bindingprotein 48 Terminase large subunit 49 Terminase small subunit 50 CobTprotein 51 Putative chromosome segregation protein, SMC ATPasesuperfamily 52 Deoxycytidylate deaminase (EC 3.5.4.12) 53 aceE; pyruvatedehydrogenase e1 component oxidoreductase protein 54 Hypotheticalprotein 55 Hypothetical protein 56 Genomic DNA, chromosome 3, BAC clone:F1D9 57 SWF/SNF family helicase 58 Aspartic acid-rich protein aspolin259 CDEP 60 Nucleolin 61 Alpha/beta hydrolase fold:Esterase/lipase/thioesterase family . . . 62 Normocyte-binding protein 163 Hypothetical protein 64 DNA repair protein recN 65 Homeobox-leucinezipper protein 66 Hypothetical protein 67 gp56 dCTPase

Example 5 Use of a Water-Conditioning Agent in the Phage Cocktail

Phage cocktail INT-40 at a final concentration of 1×10⁷ pfu/ml wasplaced into treated (containing 50 mM citrate-phosphate-thiosulfate(CPT) buffer, comprising about 40 mg sodium thiosulfate, 6.0 gm disodiumphosphate (anhydrous), 1.1 gm citric acid (anhydrous) per liter ofdeionized water, pH 7.0 (added at a 1:10 ratio to water) and untreated(distilled water) solutions containing added bleach at the levelsindicated in Table 18, and allowed to stand for one hour at roomtemperature. Samples were taken, and 10 microliters were spotted ontoBHI agar medium containing lawns of C. perfringens ATCC 13124 andallowed to dry. Plates were incubated overnight at 37° C., and phageinactivation was scored by the absence of a lytic clearing zone visibleon the bacterial lawn.

Results: The results in Table 1 demonstrated the thiosulfate-containingbuffer was able to protect phage cocktail INT-401 against oxidation dueto chlorine bleach exposure. This conditioning agent could therefore beapplied to chlorinated water as a means to allow the phage cocktail toretain activity in a commercial poultry watering system.

TABLE 18 Water Conditioning Agent Allowing Protection of Phage CocktailINT-401 in the presence of Hypochlorite Hypochlorite Concentration LysisResponse vs. ATCC 13124 (ppm) Conditioned Water Unconditioned Water 0 ++++ 0.5 ++ + 1 ++ + 2 + − 4 + − 6 + − Lysis Response: ++ Clear Lysis +Partial Lysis − No Lysis

The terms “first,” “second,” and the like, herein do not denote anyorder, quantity, or importance, but rather are used to distinguish oneelement from another, and the terms “a” and “an” herein do not denote alimitation of quantity, but rather denote the presence of at least oneof the referenced item. All ranges disclosed herein are inclusive andcombinable.

While the invention has been described with reference to a preferredembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing fromessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims.

All cited patents, patent applications, and other references areincorporated herein by reference in their entirety.

1. A method of reducing chicken mortality due to C. perfringensinfections comprising administering a purified bacteriophage preparationcomprising four or more C. perfringens-specific bacteriophage whereineach bacteriophage has lytic activity against at least five C.perfringens strains which cause necrotic enteritis in poultry.
 2. Themethod of claim 1, wherein the at least five C. perfringens strainscomprise ATCC strain 3624 and ATCC strain
 9856. 3. The method of claim1, wherein the C. perfringens strains comprise at least five of ATCCstrain 25768, ATCC strain 3624, ATCC strain 9856, ATCC strain 3628, ATCCstrain 13124, ATCC strain PTA-8495, NRRL strain B-50143, NRRL strainB-50144, NRRL strain B-50145, or a combination thereof.
 4. The method ofclaim 1, wherein the preparation has lytic activity against ATCC strain25768, ATCC strain 3624, ATCC strain 9856, ATCC strain 3628, ATCC strain13124, ATCC strain PTA-8495, NRRL strain B-50143, NRRL strain B-50144,NRRL strain B-50145.
 5. The method of claim 4, wherein the bacteriophagepreparation is incapable of infecting at least ten strains of E. coli,L. monocytogenes, S. enterica, and P. aeruginosa.
 6. The purifiedbacteriophage preparation of claim 1, comprising five or more C.perfringens-specific bacteriophage, wherein each bacteriophage has lyticactivity against at least five C. perfringens strains.
 7. The method ofclaim 6, wherein the at least five C. perfringens strains comprise ATCCstrain 3624 and ATCC strain
 9856. 8. The method of claim 6, wherein theC. perfringens strains comprise at least five of ATCC strain 25768, ATCCstrain 3624, ATCC strain 9856, ATCC strain 3628, ATCC strain 13124, ATCCstrain PTA-8495, NRRL strain B-50143, NRRL strain B-50144, NRRL strainB-50145, or a combination thereof.
 9. The method of claim 6, wherein thepreparation has lytic activity against ATCC strain 25768, ATCC strain3624, ATCC strain 9856, ATCC strain 3628, ATCC strain 13124, ATCC strainPTA-8495, NRRL strain B-50143, NRRL strain B-50144, NRRL strain B-50145.10. The method of claim 9, wherein the bacteriophage preparation isincapable of infecting at least ten strains of E. coli, L.monocytogenes, S. enterica, and P. aeruginosa.
 11. The method of claim1, comprising CPAS-12 (accession number PTA-8479), CPAS-15 (accessionnumber PTA-8480), CPAS-16 (accession number PTA-8481) and CPLV-42(accession number PTA-8483).
 12. The method of claim 6, comprisingCPAS-12 (accession number PTA-8479), CPAS-15 (accession numberPTA-8480), CPAS-16 (accession number PTA-8481), CPLV-42 (accessionnumber PTA-8483) and CPAS-7 (accession number PTA-8482).
 13. The methodof claim 1, wherein the bacteriophage preparation lyses greater than orequal to 85% of at least 40 screened C. perfringens strains, and whereinthe bacteriophage preparation is incapable of infecting at least tenstrains of E. coli, L. monocytogenes, S. enterica, and P. aeruginosa.14. The method of claim 13, wherein the bacteriophage preparation lysesgreater than or equal to 85% of at least 45 screened C. perfringensstrains.
 15. The method of claim 14, wherein each of the individual C.perfringens-specific bacteriophage lyses 15% to 90% of the screened C.perfringens strains.
 16. The method of claim 1, further comprising apharmaceutically acceptable excipient.
 17. The method of claim 16,wherein the pharmaceutically acceptable excipient is awater-conditioning agent.
 18. The method of claim 17, wherein thewater-conditioning agent is a 50 mM citrate-phosphate-thiosulfate buffercomprising about 40 mg sodium thiosulfate, 6.0 gm disodium phosphate(anhydrous), 1.1 gm citric acid (anhydrous) per liter of deionizedwater, pH 7.0, added at a 1:10 or greater ratio.
 19. The method of claim18, wherein the administration of the purified bacteriophage preparationcomprises administration in water at temperatures of up to 50° C. 20.The method of claim 20, wherein the administration
 21. A method ofselecting a C. perfringens host strain suitable for propagatingbacteriophage from a plurality of C. perfringens test strainscomprising: identifying one test strain from the plurality of teststrains as a C. perfringens species that produces disease in poultry toproduce a confirmed strain; and identifying confirmed strains thateither have minimal antibiotic resistance or that have an absence ofanimal-virulence markers other than those for C. perfringens therebyidentifying the C. perfringens host strain suitable to propagatebacteriophage.
 22. A method for selecting bacteriophage for use intreating disease caused by C. perfringens comprising: isolatingbacteriophage from environmental sources; producing two or more inoculumby inoculating and incubating the isolated bacteriophage with two ormore phylogenetically distinct C. perfringens strains wherein eachphylogenetically distinct C. perfringens strain is inoculated andincubated with the isolated bacteriophage in a separate container;plating the inoculum to produce plaques; and picking the individualplaques to select bacteriophage for use in treating disease caused by C.perfringens.
 23. A method for reducing chicken mortality due to C.perfringens infections comprising: isolating bacteriophage fromenvironmental sources; producing two or more inoculum by inoculating andincubating the isolated bacteriophage with two or more phylogeneticallydistinct C. perfringens strains wherein each phylogenetically distinctC. perfringens strain is inoculated and incubated with the isolatedbacteriophage in a separate container; plating the inoculum to produceplaques; picking the individual plaques to select bacteriophage for usein treating disease caused by C. perfringens; growing up thebacteriophage from the individual plaques; combining the bacteriophageto produce a bacteriophage preparation comprising four or more C.perfringens-specific bacteriophage wherein each bacteriophage has lyticactivity against at least five C. perfringens strains which causenecrotic enteritis in poultry; and treating chickens infected with C.perfringens with the bacteriophage preparation.